HIV-1 Vpr plays a pivotal role in viral pathogenesis, as its functions are being linked to nuclear transport of viral pre-integration complex, viral replication and suppression of human immune function. However, little is known about the molecular mechanisms underlying these effects. In this proposal, we will focus on studying two related effects of Vpr on cell cycle G2/M control and proteolysis, which will help us to further understand the molecular basis of these viral effects on the host cellular functions. With support of the NIH R29 First Award, we have successfully accomplished the three proposed Specific Aims, i.e., 1) to define the functional domains of Vpr responsible for nuclear localization, G2 arrest and cell killing, 2) to identify the cellular pathways affected by Vpr when it interrupts the cell cycle, and 3) to investigate the specific role of PP2A in Vpr-induced G2 arrest. We showed that Vpr activities in fission yeast cells are very similar to those in mammalian cells. We also found that Vpr does not induce G2 arrest through the classic DNA damage or replication checkpoints but instead uses an alternative PP2A-mediated regulatory pathway. In addition, we have identified a number of genes which when overexpressed suppress the G2 arrest and nuclear localization of Vpr, and these suppressors led us to uncover a new role for Vpr in the regulation of proteolysis. For the proposed studies, we hypothesize that Vpr induces G2 arrest through a novel PP2A-mediated regulatory pathway(s), and Vpr affects proteolysis by interaction with the proteasome on the nuclear periphery. Three new specific aims are proposed to test these hypotheses. 1) To define and characterize the cellular components of the new PP2A-mediated regulatory pathway by which Vpr induces G2 arrest. 2) To test the potential effect of Vpr on proteasome-related activities including proteolysis and MHC class I antigen processing and presentation. 3) To investigate biological significance of the Vpr-HHR23A interaction and its specific role in proteolysis. The proposed studies combine the use of biochemical and genetic approaches in both mammalian and fission yeast model system, which should yield important new insights into fundamental aspects of the effect of Vpr on these two basic cellular functions.